Low Molecular Weight Protein Nephrotoxicity Significant advances have been made over the past twenty-five years in the characterization of tubulointerstitial renal injury caused by immunoglobulin free light chains (abbreviated FLC in this application) and in elucidation of the structure-function relationships involved in FLC-mediated tubular damage. The molecular pathogenesis has been described in some detail, increasing the potential to create novel treatments designed to interrupt pathological proceses and prevent progresive los of renal function related to deposition of monoclonal FLCs. Oxidative stres is a critical determinant of cell function and fate, especially in disease states. Published and preliminary studies from this laboratory demonstrated that endocytosis of FLCs generates hydrogen peroxide and induces intracellular oxidative stress that promotes renal epithelial cell activation and injury. The first aim will characterize further the effects of FLC on proximal tubular epithelial cell signaling events that lead to apoptosis or cell survival. The seminal observations that demonstrated the mechanism and functional significance of intraluminal cast formation have been published by this laboratory, and recent unpublished studies have provided critical proof of concept experiments that cast formation causes acute kidney injury and can be prevented with the use of a competitor peptide that binds to the FLC binding domain (LCBD) on Tamm-Horsfall protein (THP). These studies permit the singular focus of the second aim on the interactions of FLC with the LCBD on THP as the proximate cause of cast nephropathy. The overarching theory of this application is that tubulointerstitial renal damage from FLC is a complex process that involves both activation and injury of proximal tubular cells and intraluminal cast formation. These events can occur independently or synergize to produce the clinical syndromes of acute kidney injury and chronic kidney disease. Two aims will be pursued: Aim 1. Define the intracellular signaling events that are altered and effect cell survival following exposure of proximal tubular cells to FLC. The specific hypothesis to be tested is that endocytosis of FLCs into the proximal tubule activates c-Src (60-kDa product of c- src, also known as pp60c-src) and apoptosis signal-regulating kinase 1 (ASK1) signaling kinases, which activate downstream molecules that work in concert to balance cell survival and apoptosis. Aim 2. Define the molecular interaction between FLCs and the FLC binding domain (LCBD) on THP and the functional significance of this interaction. The hypothesis to be tested is that small molecular weight nonpeptide and peptide compounds effectively compete with FLC binding to the LCBD on THP and prevent cast nephropathy. The long-term goal of this project - understanding the molecular mechanisms involved in these processes - offers the potential to develop new treatments designed to ameliorate renal failure in the setting of overproduction of monoclonal FLC, such as multiple myeloma. ! PUBLIC HEALTH RELEVANCE: Multiple myeloma constitutes 12-13% of hematologic malignancies in the US and is a significant problem in the veteran population. The incidence of renal dysfunction, which contributes to morbidity and mortality in myeloma, is almost 50% at presentation. This laboratory is dedicated to defining the role of immunoglobulin free light chains in acute kidney injury and in chronic kidney disease. This application will build upon previous findings and proposes a series of experiments that will define the mechanism and consequences of generation of the intrarenal pro-inflammatory and pro-apoptotic responses and will further elucidate the mechanism of cast nephropathy. The proposal offers the potential to develop new treatment modalities to ameliorate renal failure especially in patients with multiple myeloma. !